This invention relates to an apparatus and method of use for a new simply constructed bioreactor made at least partially of gas permeable materials. The bioreactor is useful for culturing cells and tissues in suspension in a liquid nutrient medium with minimum turbulence. The bioreactor may include ports for easy access to the vessel culture, allowing the growth substrate to be varied for optimum performance.
A primary use is in research where large numbers of cells are grown to refine the minute quantities of an active material (e.g., proteins) that the cells might secrete. Another use of bioreactors is the scale-up of laboratory cell culture processes for commercial purposes to mass produce the active proteins made by genetically engineered cells. Because of the need to culture mammalian cells in the laboratory in large quantities, bioreactors have become an important tool in research and production of cells that produce active proteins.
A current problem in tissue culture technology is the unavailability of an inexpensive bioreactor for the in vitro cultivation of cells and explants that allows easy access to the materials contained in the vessel. Several devices presently on the market have been used with considerable success, but each has its limitations which restrict usefulness and versatility.
Cell culturing devices range upward in complexity from the petri dish, to plastic flasks, to sophisticated computer controlled bioreactors. In the past, manufacturers have promoted various technologies to culture cells in the laboratory. Simple adaptations of fermentors (stirred tanks) used for the culture of bacteria were marketed previously as the answer to culturing delicate mammalian cells. One of the principal factors limiting the performance of these systems is their inability to minimize turbulence due to stirring, i.e., shear due to fluid flow, and hence preventing free form association of cells in three dimensions.
Another utilized technology is microcarrier cell culture, which involves the use of substrate particles, generally collagen-coated beads, to culture anchorage dependent cells. Bioreactors for microcarrier or suspension cells must suspend the cells and substrate in a fluid medium. In the past, this generally was done with an impeller in a stirred tank. Oxygen was provided by sparging (i.e., bubbling) air through the liquid medium. Both the impeller and the bubbling air, unfortunately, create turbulence.
In recent years, a variety of devices have been designed involving horizontal rotating vessels for the suspension of solids in liquid slurries, including bioreactors for cell culture. The primary inventor of the present invention was a co-inventor on six prior patents involving bioreactor systems or methods. They are as follows: U.S. Pat. No. 5,155,035, Schwarz, et al., "Method For Culturing Mammalian Cells In A Perfused Bioreactor" issued Oct. 13, 1992; U.S. Pat. No. 5,155,034, Wolf, et al., "Three-Dimensional Cell To Tissue Assembly Process" issued Oct. 13, 1992; U.S. Pat. No. 5,153,133, Schwarz, et al., "Method For Culturing Mammalian Cells In A Horizontally Rotated Bioreactor", issued Oct. 6, 1992; U.S. Pat. No. 5,153,131, Wolf, et al., "High Aspect Vessel And Method Of Use", issued Oct. 6, 1992; U.S. Pat. No. 5,026,650, Schwarz, et al., "Horizontally Rotated Cell Culture System With A Coaxial Tubular Oxygenator", issued Jun. 5, 1991; and U.S. Pat. No. 4,988,623, Schwarz, et al., "Rotating Bio-Reactor Cell Culture Apparatus", issued Jan. 29, 1991. These patents are incorporated herein by reference as if set out fully verbatim. U.S. Pat. No. 5,153,132, Goodwin, et al., "Three-Dimensional Co-Culture Process", issued Oct. 6, 1992, is closely related to this group of patents, and is also incorporated herein fully as if set out verbatim.
These prior patents disclose apparatuses that use either an internal cylindrical oxygenator or a flat disk shaped oxygenator membrane inserted internally between two pieces of the vessel. Both types of vessels require oxygen injectors.
Specifically, U.S. Pat. Nos. 4,988,623; 5,153,133; and 5,155,034 disclose culture vessels, allowing three-dimensional cell growth, that are shaped similarly to each other due to a central tubular member that functions as a membrane to allow air to be injected through the tubular membrane and into the fluid medium. These patents also disclose internal circularly disposed sets of blade members that rotate around the central horizontal axis, to move the fluid medium within the culture vessel. The apparatus disclosed in U.S. Pat. No. 5,026,650 is similar to the first three patents, but does not contain the blades that move the fluid medium.
U.S. Pat. No. 5,153,131 discloses a bioreactor vessel without mixing blades or a central tubular membrane. This apparatus still requires injection of air into the bioreactor vessel. Air travels through an air inlet passageway, through a support plate member, through a screen and filter cloth, and through a flat disk permeable membrane wedged between the two sides of the vessel housing. The construction of either the tubular membrane or the flat disk one is expensive in terms of the raw materials used (silicone rubber) and the manufacturing process. These membranes are delicate, difficult to install, and require servicing and testing to insure they are free of leaks. A single small hole in the membrane admits air in minute quantities, which forms into air bubbles. As mentioned above, even one air bubble causes damaging turbulence which inhibits or prevents cell growth.
Furthermore, the flat disk membrane in U.S. Pat. No. 5,153,131 flexes to cause mixing(col. 8,line 63 to col. 9, line 5), which is stated to be critical for the distribution of air throughout the culture media. This mixing effect, however, disrupts three dimensional cell growth. The simulation of the zero gravity environment that this design supposedly offers is incomplete because membrane flexing would not occur in a truly zero gravity environment, such as aboard the Space Shuttle in orbit around the earth. Consequently, an improved method of suspending particles(cells and their substrate) that minimizes fluid turbulence, while at the same time providing the required oxygen transfer, is needed to improve the performance of bioreactors. It is an object of the present invention to provide both an apparatus and a method for culturing cells that overcomes the technological limitations of prior bioreactor systems.